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OBJECTIVES: To design a reliable model in the context of prenatal screening for assigning the risk in an individual pregnancy of Smith-Lemli-Opitz syndrome (SLOS) and assess its performance. SETTING: A 2nd trimester screening programme for Down's syndrome that measures unconjugated estriol (uE3) along with other serum markers. METHODS: Development of individual risk estimates with a trivariate model incorporating measurements of maternal serum uE3, alpha-fetoprotein (AFP), and human chorionic gonadotropin (hCG) in both SLOS and unaffected pregnancies. RESULTS: Population parameters were computed for the three analytes, as were pairwise correlation coefficients and truncation limits, based on an unbiased collection of 29 affected pregnancies. Published parameters were used for unaffected pregnancies. With a cut off level of risk of 1:50, 62% of SLOS pregnancies can be detected by initially identifying 0.34% of unaffected pregnancies as screen positive. About 1 in 90 screen positive pregnancies will be affected. CONCLUSIONS: It is possible to screen for SLOS as an add on to existing 2nd trimester maternal serum screening, if uE3 is already being measured. A large, prospective trial is necessary to determine whether diagnostic testing can be performed in maternal urine or serum rather than amniotic fluid.

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Most clinical conditions are accompanied by corresponding changes in serum levels of some, if not all, of the acute phase proteins. While conditions that affect the acute phase proteins are usually inflammatory in nature, non-inflammatory conditions also can cause changes (e.g., malnutrition, some malignancies without secondary inflammation, and genetic polymorphism). Only after the confounding effects of non-inflammatory conditions are taken into account can these measurements be used to detect and stage the inflammatory process and to evaluate the impact of treatment. In this third article in a series, reference ranges for serum levels for three of the acute phase proteins that increase during inflammation are examined: alpha1-acid glycoprotein (orosomucoid), alpha-antitrypsin, and haptoglobin. The study is based on a cohort of 55,199 Caucasian individuals from northern New England, tested in our laboratory between 1994 and 1999. Measurements were standardized against CRM 470 (RPPHS) and analyzed using a previously described statistical approach. Individuals with unequivocal laboratory evidence of inflammation (C-reactive protein of 10 mg/l or higher) were excluded. Levels of a,-acid glycoprotein changed little during life and between the sexes. Levels of alpha1-antitrypsin varied somewhat by age, rising slightly beyond age 55; males followed a pattern similar to that for females. For this protein, it was necessary to apply two equations to describe the lower levels associated with certain phenotypes. Haptoglobin levels fell significantly during the first decade of life for both males and females and climbed thereafter. Males and females displayed a similar pattern. When values were expressed as multiples of the age- and gender-specific median levels, the resulting distributions fitted a log-Gaussian distribution well over a broad range. When patient data are normalized in this manner, the distribution parameters can be used to assign a centile corresponding to an individual's measurement, thus simplifying interpretation.

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The relationship between atheroma lipid composition and serum lipoprotein and oxidation measurements has not been fully explored. To address this question, we studied serum, plasma, and aortic wall specimens from 66 subjects undergoing coronary artery bypass graft surgery. The lipid composition of aortic specimens was characterized in terms of cholesterol ester and cholesterol crystal plus phospholipid by using hot-stage polarizing light microscopy; tissue oxidation status was assessed by measuring conjugated dienes. Serum lipoprotein-related measurements included total cholesterol, triglyceride, low density lipoprotein (LDL) cholesterol, high density lipoprotein (HDL) cholesterol, apolipoproteins B and AI, and lipoprotein(a). Oxidation status was assessed by measuring LDL mobility, thiobarbituric acid-reactive substances, LDL conjugated dienes, and IgG and IgM autoantibodies against oxidized LDL. Fasting blood glucose was also determined. Lesion cholesterol crystal plus phospholipid content was associated inversely with serum HDL cholesterol levels (r=-0.279, P=0.029) and positively with fasting blood glucose (r=0.359, P=0.016), LDL mobility (0.276, P<0.05), and IgM autoantibodies against oxidized LDL (r=0.272, P=0.037). There was also a significant relationship between the level of aortic tissue conjugated dienes and plasma LDL mobility (r=0.332, P=0.007). In multivariate analysis, IgM autoantibodies against oxidized LDL, fasting blood glucose, and LDL mobility, in descending order of significance, together accounted for 35% of the variability in aortic lesion cholesterol crystal plus phospholipid content. These data support direct and independent roles for oxidation and hyperglycemia in the pathophysiology of atherosclerosis.

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We examined the effect of antigen source on an enzyme-linked immunosorbent assay (ELISA) for autoantibodies against oxidized low-density lipoprotein (LDL). Serum samples from 20 subjects with systemic lupus erythematosus (SLE) and from 20 controls were assayed for immunoglobulin G (IgG) and immunoglobulin M (IgM) autoantibodies against oxidized LDL, using either a pooled or individual (n = 3) LDL preparation as antigen. For IgG autoantibodies against oxidized LDL there was a relationship (r approximately 0.5, P < 0.01) between data obtained using individual versus pooled antigen preparations. Bias plots demonstrated consistent inverse, concentration-dependent relationships (r approximately -0.6, P < 0.001). The difference in IgG autoantibodies against oxidized LDL levels between SLE patients and controls was underestimated (39-58%) when assays used individual rather than pooled LDL antigen. For IgM autoantibodies against oxidized LDL the direct relationships were stronger (r approximately 0.8, P < 0.001) and the concentration-dependent relationships weaker (r approximately -0.3, significance variable) than for IgG autoantibodies against oxidized LDL. Variations between LDL preparations suggested that a pooled antigen would give a more stable assay. Thus, LDL antigen source is important in assays for both IgG and IgM autoantibodies against oxidized LDL.

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OBJECTIVE: To characterize the antinuclear antibody (ANA) titer distributions and patterns in normal subjects, segregated by age and sex. METHODS: Sera were obtained from 183 blood donors (130 females, 53 males) aged 20-63 years, from 200 schoolchildren (100 females, 100 males) aged 10-19 years, and from 237 children (102 females, 135 males) aged 3 months to 9 years whose sera were received for unrelated clinical testing. ANA was assayed by indirect immunofluorescence using HEp-2 cells as substrate. RESULTS: In adults, ANA titers were slightly higher in females than in males (p=0.053); there was no sex effect in subjects aged <20 years. ANA titer increased significantly with age only among females (p<0.01). Homogeneous staining was associated with lower titers than speckled or nucleolar staining (p=0.058), at least in part because of antigen density in the test substrate itself. The frequency of cytoskeletal staining decreased (p<0.01) with age, while that of nucleolar staining increased (p<0.01). CONCLUSION: Reference ranges for ANA vary by age, sex, and immunofluorescence pattern. Therefore, all these variables must be considered in the interpretation of ANA results.

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Inflammation is associated with diverse clinical conditions accompanied by characteristic changes in serum levels of the acute-phase proteins that can be used to stage the inflammatory process and evaluate the impact of treatment. Some acute-phase proteins increase during inflammation, while others, such as albumin, transferrin, and transthyretin, decrease. The current study reports reference ranges for serum levels of albumin, transferrin, and transthyretin based on a cohort of over 124,000 Caucasian individuals from northern New England, tested in our laboratory between 1986 and 1998. Measurements were standardized against CRM 470 (RPPHS) and analyzed using a previously validated statistical approach. Individuals with laboratory evidence of inflammation (C-reactive protein of 10 mg/L or higher) were excluded. The levels of all three analytes varied by age, generally rising until the second or third decade of life and then decreasing thereafter. Albumin and transthyretin levels were higher during midlife among males as compared to females; the maximum being at 25 years for albumin (5%) and 35 years for transthyretin (16%). In contrast, above the age of 10 years, transferrin levels were increasingly higher among females (7% at 20 years). When values were expressed as multiples of the age- and gender-specific median levels, the resulting distributions fitted a log-Gaussian distribution. When patient data are normalized in this manner, the distribution parameters can be used to assign a corresponding centile to an individual's measurement simplifying interpretation. The ultimate interpretation of an individual's measurement relies upon the clinical setting.

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Serum immunoglobulins are measured millions of times each year, yet clinical interpretations remain hampered by inadequate age- and gender-specific reference limits. In order to provide more reliable and comprehensive reference distributions for IgA, IgG, and IgM measurements, we analyzed automated immunoassay values from 115,017 serum samples from northern New England patients (99% Caucasian) who were tested in our laboratory between 1986 and 1995. Measurements were standardized to reference material, CRM 470 (RPPHS). A simple, practical, and clinically relevant approach was used to determine reference distributions for the immunoglobulins over a wide range of ages for males and females. Levels of IgA and IgM varied considerably by age, and by gender for IgM. For each of the analytes, the observed 5th and 95th centiles were symmetric about the median and approximately constant over the entire age range. When immunoglobulin reference values are expressed as multiples of the age- and gender-specific regressed medians, the resulting distributions fit a log-Gaussian distribution well. This finding enables interpretation of serum immunoglobulin measurements using a common unit (multiples of the median) that is independent of age or gender. Insights gained from this study can help improve and simplify the interpretation of immunoglobulin measurements.

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Although in vitro studies support a pathophysiologic role for lipoprotein(a) [Lp(a)] in the development of atherosclerosis, and retrospective studies consistently report that there is a relationship between Lp(a) and ischemic heart disease (IHD), the conclusions drawn from prospective studies about this relationship have been inconsistent. To address this issue, we have performed a metaanalysis of data available from prospective studies. Lp(a) concentrations expressed as mass units vary markedly between studies, reflecting the need for assay standardization. In 12 of 14 prospective studies, Lp(a) concentrations are higher in subjects who later develop IHD (cases) than in those who do not (controls), although there is variation in the size of the effect. Sample storage temperature may contribute to this variability. When the studies are analyzed collectively, Lp(a) concentrations are significantly higher in cases than in controls, and the extent of the effect is similar in men and women. These findings provide evidence in support of a causal role for Lp(a) in the development of atherosclerosis. Measurement of Lp(a) may be useful to guide management of individuals with a family history of IHD or with existing disease. The separation in values between cases and controls is not, however, sufficient to allow the use of Lp(a) as a screening test in the general population.

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To examine the relationship between apolipoprotein E and serum oxidation status, we assayed apolipoprotein E level, apolipoprotein E phenotype, and levels of lipid peroxides and transition metal ions and their binding proteins in sera from apparently healthy individuals. The study group included 129 women aged 22-63 years and 53 men aged 22-56 years. Among subjects with apolipoprotein E 4/3 phenotype, lipid peroxide levels were higher compared with E 3/2 phenotype (786 +/- 182 nmol/l vs. 659 +/- 174 nmol/l, P = 0.015), and ceruloplasmin levels were slightly higher compared with apolipoprotein E 3/3 phenotype (0.28 +/- 0.08 mg/l vs. 0.26 +/- 0.06 mg/l, P = 0.035). In the study group as a whole, there were significant associations between serum apolipoprotein E level, and serum levels of ceruloplasmin (r = 0.266, P < 0.001) and ferritin (r = 0.2, P < 0.007). Among subjects with apolipoprotein E 4/3 phenotype, there was a significant association between serum apolipoprotein E and lipid peroxide levels (r = 0.470, P < 0.01), which was not apparent among subjects with E 3/3 or E 3/2 phenotypes. In multivariate analysis, apolipoprotein E phenotype was a small but significant independent contributor to variation in serum lipid peroxide levels. These data suggest that there may be heterogeneity among apolipoprotein E phenotypes in their relationships with serum lipid oxidation status.

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We recently demonstrated that insulin growth factor-I (IGF-I) cosegregates with bone mineral density (BMD) in progenitor crosses of two inbred strains of mice. Additionally, we reported that men with idiopathic osteoporosis (IOM) have low serum IGF-I levels, which can be related to BMD and bone turnover. In this study, we considered the possibility that serum IGF-I levels are influenced by molecular genetic variation in the IGF-I structural gene, and that a polymorphic microsatellite (CA repeat) in this locus can be used as a genetic marker for such comparisons. We studied 171 men and women, classified according to the trial in which they were participating. First, in 25 Caucasian men with IOM we noted a very high frequency (64%) of homozygosity for the most common allele (192 bp) in a dinucleotide repeat 1 kb upstream from the transcription start site of the IGF-I gene. This compared with a frequency of only 32% in healthy populations (both men and women) (P < 0.004). Next, we determined that for 116 healthy Caucasian men and women the 192/192 genotype was associated with lower serum IGF-I levels than all other genotypes (192/192: 129 +/- 7 ng/mL vs. others: 154 +/- 7 ng/mL, P = 0.03). We also noted that subjects possessing one 194-bp allele exhibited serum IGF-I levels 25% higher than those homozygous for 192 bp (192/192), (P < 0.005) even after correction for age and sex. Similarly, for men with the 192/192 genotype, serum IGF-I concentrations were lower than any other genotype (145 +/- 10 ng/mL vs. 183 +/- 9 ng/ml P < 0.02). In conclusion, low serum IGF-I levels found in men with IOM are associated with homozygosity for a specific allele of the IGF-I microsatellite (192/192), and individual variation in serum IGF-I levels is influenced by genetic factors and may be specifically influenced by variation at the IGF-I structural locus. Further family and pedigree studies are needed to characterize the relationship of bone mass acquisition to the IGF-I genotype.

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BACKGROUND: We aimed to determine the relationship between ruptured abdominal aortic aneurysm (AAA) and serum concentrations of lipids and apolipoproteins. METHODS: A cohort of 21 520 men, aged 35-64 years, was recruited from men attending the British United Provident Association (BUPA) clinic in London for a routine medical examination in 1975-1982. Smoking habits, weight, height and blood pressure were recorded at entry. Lipids and apolipoproteins were measured in stored serum samples from the 30 men who subsequently died of ruptured AAA and 150 matched controls. RESULTS: Triglyceride was strongly related to risk of ruptured AAA. In univariate analyses the risk in men on the 90th centile of the distribution relative to the risk in men on the 10th (RO10-90) was 12 (95% confidence interval [CI] : 3.8-37) for triglyceride, 5.5 (95% CI: 1.8-17) for apolipoprotein B (apoB) (the protein component of low density lipoprotein [LDL]), 0.15 (95% CI : 0.04-0.56) for apo A1 (the protein component of high density lipoprotein [HDL]), 3.7 (95% CI: 1.4-9.4) for body mass index and 3.0 (95% CI: 1.1-8.5) for systolic blood pressure. Lipoprotein (a) (Lp(a)) was not a significant risk factor (RO10-90 = 1.6, 95% CI: 0.6-3.0). In multivariate analysis triglyceride retained its strong association. CONCLUSION: Triglyceride appears to be a strong risk factor for ruptured AAA, although further studies are required to clarify this. If this and other associations are cause and effect, then changing the distribution of risk factors in the population (by many people stopping smoking and adopting a lower saturated fat diet and by lowering blood pressure) could achieve an important reduction in mortality from ruptured AAA.

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Recent guidelines established by the Association of State and Territorial Public Health Laboratory Directors (ASTPHLD) and the U.S. Centers for Disease Control and Prevention (CDC) recommend the use of a two-test protocol for the serologic diagnosis of Lyme disease (LD). The two-test protocol relies on a sensitive screening test, which is followed by specific immunoglobulin M (IgM) and/or IgG immunoblotting (IB), depending on the date of disease onset, of all samples with equivocal and positive screening test results. We evaluated a commercially available IgM-IgG enzyme-linked immunosorbent assay (ELISA) and separate IB tests for IgM and IgG antibodies to Borrelia burgdorferi as candidate assays for the two-test protocol. Serum samples obtained from healthy controls (n = 29), from patients with diagnoses or laboratory findings associated with serologic cross-reactivity to LD (n = 24), and from patients with well-documented early- and late-stage LD provided by the CDC and the College of American Pathologists (n = 53) were examined to determine each assay's individual sensitivity and specificity. No false-positive results were detected among the healthy controls by either ELISA or IB, whereas four false-positive ELISA results were recorded within the cross-reactive group. None of these sera, however, were positive for either IgM or IgG reactivity according to IB band criteria. With regard to the patients with LD, we determined the sensitivity and specificity of the ELISA to be 96 and 100%, respectively, compared with the reference data provided for these specimens. When we compared our IB results with data from CDC, the assay sensitivity and specificity were 80 and 96.2%, respectively, for IgM and 81.8 and 95.8%, respectively, for IgG. Pursuant to this evaluation we assessed the suitability of the two-test protocol by performing a retrospective analysis using clinical history to define samples as positive or negative for LD. We determined clinical sensitivity and specificity for all study subjects (n = 112) to be 50 and 100%, respectively. A reduction in the clinical sensitivity of the two-test protocol was associated with a lack of antibody response or seroconversion in LD patients treated with antibiotics. We conclude that the CDC-ASTPHLD guidelines provide useful criteria for test performance and interpretation aimed at standardizing the serologic diagnosis of LD.

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IgG autoantibodies against malondialdehyde-modified LDL (alpha oxLDL), antiphospholipid antibodies (APA) and oxidation- and lipoprotein-related analytes were assayed in sera from healthy subjects (51 males, 115 females, aged 22-63 years). alpha OxLDL levels were associated (P < 0.03) with IgG alpha cardiolipin (r = 0.18), IgM alpha cardiolipin (r = 0.17) and IgM alpha phosphatidyl-serine (r = 0.16) but not with age, cholesterol, triglyceride, apolipoproteins B and AI, lipoprotein(a), lipid peroxides, ceruloplasmin, copper, ferritin, transferrin or iron. APA levels were inversely associated with levels of both oxidation- and lipoprotein-related analytes. Ferritin (3.5%) and alpha oxLDL (1.4%) contributed independently to variation in IgG alpha cardiolipin levels, and apo B (2%) to variation in IgM alpha cardiolipin levels. These associations are small, indicating that there are no major biological associations between the measured variables. The lack of association between alpha oxLDL and lipoprotein- or oxidation-related analytes suggests that the relevant antigen is not in serum.

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STUDY OBJECTIVE: To determine the efficacy of high-dose ascorbate supplementation in lowering lipoprotein(a) [Lp(a)] levels in patients with premature coronary heart disease (CHD). DESIGN: Randomized, double-blind, placebo-controlled trial. SETTING: Outpatient clinic. PATIENTS: Forty-four patients with documented premature CHD, defined as confirmed myocardial infarction and/or angiographically determined stenosis of 50% or greater in at least one major coronary artery before age 60 years. INTERVENTIONS: Patients were block randomized on the basis of age, gender, and screening Lp(a) concentrations to receive ascorbate 4.5 g/day or placebo for 12 weeks. MEASUREMENTS AND MAIN RESULTS: High-dose ascorbate was well tolerated and produced a marked elevation in mean plasma ascorbate levels (+1.2 mg/dl; p < 0.001). Multiple linear regression analysis revealed no significant effect of supplementation on postintervention Lp(a) levels (p = 0.39) in a model that included treatment group assignment, and baseline Lp(a) levels. CONCLUSIONS: Our findings do not support a clinically important lowering effect of high-dose ascorbate on plasma Lp(a) in patients with premature CHD.

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The relations between oxidation-related analytes and lipoprotein risk factors for coronary heart disease are poorly understood. To address this issue, ceruloplasmin, copper, iron, ferritin, cotinine, lipid peroxides, cholesterol, triglyceride, apoB, apoA-I, and lipoprotein(a) levels were measured in sera from apparently healthy subjects (51 men and 115 women). Pairwise comparisons revealed strong positive associations (P < .001) of copper and ceruloplasmin with lipid peroxides, total cholesterol, triglycerides and apoB, of transferrin with apoA-I and cholesterol, and of ferritin with triglycerides. Serum levels of oxidation-related analytes did not differ between smokers and nonsmokers. In multivariate analysis, serum copper was the major independent determinant of serum lipid peroxide level, accounting for 15% of the variability in concentration (ferritin accounted for 1.6%). Copper and ceruloplasmin accounted for 20.5% of the variation in triglyceride levels; triglycerides and apoB accounted for 12% of the variability in ferritin levels; apoB and apoA-I accounted for 9% of the variability in transferrin levels. The data suggest that serum copper contributes to lipid peroxidation in vivo. There are significant associations between lipoprotein and transition metal-related analytes, and further work is needed to elucidate the physiological basis for these relations.

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